U.S. patent application number 14/647401 was filed with the patent office on 2015-10-29 for optical microscope, and autofocus device for optical microscope.
The applicant listed for this patent is OSAKA UNIVERSITY. Invention is credited to Yoshiyuki ARAI, Takeharu NAGAI.
Application Number | 20150309298 14/647401 |
Document ID | / |
Family ID | 50776112 |
Filed Date | 2015-10-29 |
United States Patent
Application |
20150309298 |
Kind Code |
A1 |
NAGAI; Takeharu ; et
al. |
October 29, 2015 |
OPTICAL MICROSCOPE, AND AUTOFOCUS DEVICE FOR OPTICAL MICROSCOPE
Abstract
Provided is an optical microscope capable of maintaining an
objective lens in a focus state without adversely affecting a
captured image even when a substance with a weak emission is used
as an observation target object. The optical microscope includes an
observation optical system capable of capturing, with an image
pickup device (10), an optical image of an observation target
object (1) that has been obtained with an objective lens (5), and
sending out the optical image as an image signal; and an autofocus
device that adjusts a focal position of the objective lens based on
autofocus light emitted from a focusing light source (11) so as to
focus on the observation target object, wherein the autofocus light
is emitted from the focusing light source during a non-capturing
period during which the image pickup device is not capturing an
image of the observation target object.
Inventors: |
NAGAI; Takeharu; (Suita-shi,
Osaka, JP) ; ARAI; Yoshiyuki; (Suita-shi, Osaka,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OSAKA UNIVERSITY |
Osaka |
|
JP |
|
|
Family ID: |
50776112 |
Appl. No.: |
14/647401 |
Filed: |
November 20, 2013 |
PCT Filed: |
November 20, 2013 |
PCT NO: |
PCT/JP2013/081259 |
371 Date: |
May 26, 2015 |
Current U.S.
Class: |
359/363 |
Current CPC
Class: |
G02B 21/245 20130101;
G02B 21/365 20130101; G02B 21/16 20130101; G02B 21/0004 20130101;
G02B 7/28 20130101; G02B 21/02 20130101 |
International
Class: |
G02B 21/36 20060101
G02B021/36; G02B 7/28 20060101 G02B007/28; G02B 21/16 20060101
G02B021/16; G02B 21/02 20060101 G02B021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 26, 2012 |
JP |
2012-257426 |
Claims
1. An optical microscope comprising: an observation optical system
capable of capturing, with an image pickup device, an optical image
of an observation target object that has been obtained with an
objective lens, and sending out the optical image as an image
signal; and an autofocus device that adjusts a focal position of
the objective lens based on autofocus light emitted from a focusing
light source so as to focus on the observation target object,
wherein the autofocus light is emitted from the focusing light
source during a non-capturing period during which the image pickup
device is not capturing an image of the observation target
object.
2. The optical microscope according to claim 1, wherein a capturing
period during which an image of the observation target object is
captured and the non-capturing period are repeated in the image
pickup device, and the image pickup device continuously captures an
image of the observation target object during the capturing period,
while the autofocus device performs an autofocus operation during
the non-capturing period.
3. The optical microscope according to claim 2, wherein the
autofocus device selects a predetermined non-capturing period from
among a plurality of the non-capturing periods, and performs the
autofocus operation only in the selected non-capturing period.
4. The optical microscope according to claim 1, wherein the image
pickup device includes a CCD image sensor as an image pickup
element, and the non-capturing period is a readout time during
which the image pickup element transfers captured image data.
5. The optical microscope according to claim 1, wherein the
autofocus light emitted by the focusing light source is infrared
light outside the visible range.
6. The optical microscope according to claim 1, wherein the optical
microscope is used for chemiluminescence observation in which a
luminescent material that emits light by a chemical reaction is
observed as the observation target object.
7. The optical microscope according to claim 1, comprising a focus
adjustment lens that performs autofocus on an observation target
portion located in a position spaced away by a predetermined
distance from an interface of a glass substrate on which the
observation target object is placed, by using the autofocus light
reflected at the interface of the glass substrate.
8. The optical microscope according to claim 1, wherein the optical
microscope is an inverted microscope in which the objective lens is
located below the observation target object.
9. An autofocus device for an optical microscope that is used in an
optical microscope including an image pickup device capable of
sending out, as an image signal, an optical image of an observation
target object that has been obtained with an objective lens, and
that adjusts a focus position of the objective lens based on
reflected light of autofocus light emitted from a focusing light
source, wherein the autofocus light is emitted from the focusing
light source during a non-capturing period during which the image
pickup device is not capturing an image of the observation target
object.
Description
TECHNICAL FIELD
[0001] The present disclosure relates to an optical microscope
capable of sending, as an image signal, an optical image of an
observation target object that has been captured by an image pickup
device, and particularly to an optical microscope including an
autofocus device that performs focus adjustment of the objective
lens by using autofocus light, and an autofocus device for the
optical microscope.
BACKGROUND ART
[0002] Optical microscopes and their peripheral devices, which
contribute to studies in various fields, have been provided with
the function of capturing images of, in particular, an observation
target object using an image pickup device in recent years. They
send out image data of a captured still image or moving image to
external devices such as a computer and a large monitor, and are
used for image analysis and phenomenon analysis. Further, the
automation of the overall function of the optical microscope is
also progressing. For example, in order to adjust the optical
microscope while analyzing an image obtained with the optical
microscope, the positional adjustment of a stage on which the
observation target object is placed, and the operation of switching
between a plurality of mounted objective lens having different
magnifications, can be performed by a remote operation.
[0003] For example, in the field of life science studies,
time-lapse observation is often performed in which a
high-resolution analysis image is observed over a long period of
time in order to analyze the change in the microstructure inside a
cell, the pattern of cell differentiation, and the like by using a
high-resolution optical microscope. An optical microscope has a
very narrow focal length because of its high magnification, and the
observation target object will be out of a focal position of the
objective lens due to a slight vibration or change in the ambient
temperature. In addition, in order to observe the above-described
structural change inside a cell, it is often the case that a
thermal stimulation is applied externally to a cell to be observed.
If this causes a change in the ambient temperature, the focus state
to the observation target object is impaired, resulting in the
problem that changes in the cell structure cannot be observed
accurately.
[0004] For this reason, optical microscopes equipped with an
autofocus device that can automatically maintain the focus state of
the objective lens have been developed. As an autofocus device in
the field of optical microscopes, an active autofocus method having
a high-speed tracking performance is used. This active autofocus
method causes autofocus light emitted via an objective lens to be
reflected by a reflection film provided on an interface between a
slide glass on which an observation target object is placed and a
cover glass mounted on the observation target object or by the
interface itself, and from the received reflected light, accurately
measures the interval between the objective lens and the
observation target object, and maintains this interval
constant.
[0005] In recent years, there have been proposed optical
microscopes capable of performing accurate autofocus even in the
case where the interface of the slide glass and a cell nucleus
serving as the observation target object are at positions having
slightly different intervals with the objective lens, including,
for example, a case where only a single nucleus in a cell is
observed as performed in the field of the studies of life science.
For such an optical microscope, there has been proposed a technique
by which focusing is achieved at a position shifted by a
predetermined amount from a focus position obtained by the active
autofocus method, based on data obtained by a focus operation to
the slide glass or the like by the active autofocus method and a
focus operation performed by the operator to the observation target
object by using an observation optical system (See Patent Document
1).
[0006] There has also been proposed a technique by which a focus
adjustment lens system is included in the autofocus optical system
in the active autofocus method, a focus offset amount is set by
performing a focus operation by the operator in a state in which an
autofocus operation by the active autofocus method has been
performed for the slide glass or the like, and afterward, a focus
operation by the active autofocus method is performed by the focus
adjustment lens while the focus offset amount is maintained (See
Patent Document 2).
CITATION LIST
Patent Documents
[0007] Patent Document 1: JP 2011-123518A
[0008] Patent Document 2: JP 2007-148161A
DISCLOSURE OF INVENTION
Problem to be Solved by the Invention
[0009] With the above-described conventional optical microscope,
the interval between the objective lens and the slide glass or the
like on which the observation target object is placed can be
maintained constant by the active autofocus method having a
high-speed tracking performance and accuracy. Therefore it is
possible to maintain a favorable state even if the ambient
temperature changes. As a result, in particular, an observation
based on moving images and a time-lapse observation over a long
time can be performed in a suitable manner.
[0010] In the conventional active autofocus method, infrared light
outside the visible light range is used as the focusing light
source of the autofocus optical system. This makes it possible to
avoid any practical adverse effect of the infrared light from the
focusing light source on the monitoring in the observation optical
system and images captured by the image pickup device, for example,
in an ordinary optical microscope that uses a tungsten lamp or a
halogen lamp as the irradiation light source of the observation
optical system and a fluorescence observation microscope in which
near-ultraviolet light or blue light is emitted as excitation
light.
[0011] However, in the case of performing chemiluminescence
observation, which is one method of bioimaging, the emission
luminance of the observation target object is weak as compared with
fluorescence emission observation that uses excitation light
emission, and it is therefore necessary to use a detector with a
higher sensitivity, in addition to eliminating the causes of light
loss such as a filter in the observation optical system as much as
possible. Since a faint light is detected in this case, even
infrared light, used for the conventional active autofocus method
will be disruptive in obtaining an image of the observation target
object. On the other hand, the detection of weak emission requires
a long time of exposure. However, if a long-time exposure is
performed with an optical microscope in a state in which the
autofocus device is stopped, a focus shift due to a change over
time or the like cannot be avoided, making it impossible to obtain
the desired clear captured image.
[0012] Therefore, it is an object of the present application to
solve the above-described conventional problems, and the present
invention provides an optical microscope capable of maintaining an
objective lens in a focus state without adversely affecting a
captured image even when a substance with a weak emission is used
as an observation target object, and an autofocus device that can
be used for the optical microscope and enables a favorable
chemiluminescence observation.
Means for Solving Problem
[0013] In order to solve the above-described problems, an optical
microscope disclosed in the present application includes: an
observation optical system capable of capturing, with an image
pickup device, an optical image of an observation target object
that has been obtained with an objective lens, and sending out the
optical image as an image signal; and an autofocus device that
adjusts a focal position of the objective lens based on autofocus
light emitted from a focusing light source so as to focus on the
observation target object, wherein the autofocus light is emitted
from the focusing light source during a non-capturing period during
which the image pickup device is not capturing an image of the
observation target object.
[0014] An autofocus device for an optical microscope disclosed in
the present application is an autofocus device that is used in an
optical microscope including an image pickup device capable of
sending out, as an image signal, an optical image of an observation
target object that has been obtained with an objective lens, and
that adjusts a focus position of the objective lens based on
reflected light of autofocus light emitted from a focusing light
source, wherein the autofocus light is emitted from the focusing
light source during a non-capturing period during which the image
pickup device is not capturing an image of the observation target
object.
Effects of the Invention
[0015] With the optical microscope and the autofocus device for an
optical microscope disclosed in the present application, autofocus
light is emitted during the non-capturing period during which the
image pickup device is not capturing an image of the observation
target object, and it is therefore possible to avoid possible
effects of the autofocus light on a captured image even when using
a high-sensitivity detector. Accordingly, an observation image of
an observation target object in a correctly focused state can be
obtained even when the emission luminance of the observation target
object is weak.
BRIEF DESCRIPTION OF DRAWINGS
[0016] FIG. 1 is a block configuration diagram showing a schematic
configuration of an optical microscope of a first exemplary
configuration according to the present embodiment.
[0017] FIG. 2 is a diagram showing a relationship between the
exposure timing of a camera and the irradiation timing of autofocus
light in the optical microscope according to the present
embodiment.
[0018] FIG. 3 is a block configuration diagram showing a schematic
configuration of an optical microscope of a second exemplary
configuration according to the present embodiment.
[0019] FIG. 4 show exemplary images, obtained with a conventional
optical microscope, of an observation target by fluorescence
observation and chemiluminescence observation.
[0020] FIG. 5 show exemplary images, obtained with an optical
microscope according to the present embodiment, of an observation
target of fluorescence observation and chemiluminescence
observation.
DESCRIPTION OF THE INVENTION
[0021] In a conventional active autofocus method, focus position
correction is constantly performed by continuously detecting
positions by constant irradiation with autofocus light. However, it
is not necessary to constantly perform an autofocus operation, and
the focus state to the observation target object can also be
sufficiently maintained by performing an autofocus operation
discontinuously. An optical microscope disclosed in the present
application includes: an observation optical system capable of
capturing, with an image pickup device, an optical image of an
observation target object that has been obtained with an objective
lens, and sending out the optical image as an image signal; and an
autofocus device that adjusts a focal position of the objective
lens based on autofocus light emitted from a focusing light source
so as to focus on the observation target object, wherein the
autofocus light is emitted from the focusing light source during a
non-capturing period during which the image pickup device is not
capturing an image of the observation target object.
[0022] By doing so, an image of the observation target object in a
state in which autofocus light is not emitted is captured in the
image pickup device. Accordingly, it is possible to effectively
avoid any adverse effect of the autofocus light on a captured
image.
[0023] In the optical microscope disclosed in the present
application, it is preferable that a capturing period during which
an image of the observation target object is captured and the
non-capturing period are repeated in the image pickup device, and
the image pickup device continuously captures an image of the
observation target object during the capturing period, while the
autofocus device performs an autofocus operation during the
non-capturing period. This makes it possible to avoid the effect of
a focal drift during a long period of image capturing, thus
obtaining continuous captured images of the observation target
object in a correctly focused state.
[0024] Preferably, the autofocus device selects a predetermined
non-capturing period from among a plurality of the non-capturing
periods, and performs the autofocus operation only in the selected
non-capturing period. Doing so makes it possible to discontinuously
perform an autofocus operation at necessary intervals according to,
for example, the degree of focus variations, thus effectively
avoiding undesired effects that may be exerted by the autofocus
light on a sample to be observed or on a captured image.
[0025] In the optical microscope disclosed in the present
application, it is preferable that the image pickup device includes
a CCD image sensor as an image pickup element, and the
non-capturing period is a readout time during which the image
pickup element transfers captured image data. This makes it
possible to obtain image data of the observation target object
without any effect of the autofocus light by using a commonly used
CCD image sensor as the image pickup element.
[0026] Preferably, the autofocus light emitted by the focusing
light source is infrared light outside the visible range.
[0027] Furthermore, it is preferable that the optical microscope
disclosed in the present application is used for chemiluminescence
observation in which a luminescent material that emits light by a
chemical reaction is observed as the observation target object.
Even when the observation target object has a weak emission
luminance and a high-sensitive detector is used, it is possible to
obtain an image of the observation target object that is free of
the influence from the autofocus light.
[0028] It is preferable to include a focus adjustment lens that
performs autofocus on an observation target portion located in a
position spaced away by a predetermined distance from an interface
of a glass substrate on which the observation target object is
placed, by using the autofocus light reflected at the interface of
the glass substrate. Doing so makes it possible to maintain a
favorable focus state even when an observation target portion, such
as the cell nucleus of a cell disposed in a nutrient solution, of
an observation target object is positioned away from a reference
plane of autofocus.
[0029] Furthermore, the optical microscope disclosed in the present
application may be an inverted microscope in which the objective
lens is located below the observation target object.
[0030] An autofocus device for an optical microscope disclosed in
the present application is an autofocus device that is used in an
optical microscope including an image pickup device capable of
sending out, as an image signal, an optical image of an observation
target object that has been obtained with an objective lens, and
that adjusts a focus position of the objective lens based on
reflected light of autofocus light emitted from a focusing light
source, wherein the autofocus light is emitted from the focusing
light source during a non-capturing period during which the image
pickup device is not capturing an image of the observation target
object.
[0031] By doing so, an image of an observation target object in a
state in which autofocus light is not emitted can be captured by
the image pickup device, making it possible to obtain an autofocus
device that effectively avoids an adverse effect on a captured
image.
Embodiment
[0032] Hereinafter, an embodiment of an optical microscope and an
autofocus device for an optical microscope according to the present
application will be described with reference to the drawings.
[0033] FIG. 1 is a block diagram for illustrating a schematic
configuration of an upright optical microscope in which an
objective lens is disposed above an observation target object, as a
first exemplary configuration of the optical microscope according
to the present embodiment.
[0034] In the optical microscope of the first exemplary
configuration shown in FIG. 1, an observation target object 1 is
placed on a slide glass 3 serving as a glass substrate placed on a
stage 2, and is disposed in a state in which it is sandwiched
between the slide glass 3 and a cover glass 4 that covers the
observation target object 1.
[0035] The stage 2 is movable in the X-Y direction in a plane
perpendicular to an optical axis 6 of an objective lens 5 located
above the observation target object 1. Note that the stage 2 may
also be movable in the optical axis direction of the objective lens
5 (the Z direction). In addition, the movement of the stage 2 in
the X direction and the Y direction, and in some cases, the Z
direction, may be electrically powered using a motor. In that case,
a stage control portion that controls the movement of the stage 2
may be provided, and the movement of the stage can be controlled
from a control portion 31 that performs overall control of the
optical microscope, in conjunction with the operation of other
members, for example, the operation of a focal position of the
objective lens 5.
[0036] The optical microscope of the present exemplary
configuration includes an observation optical system composed of
the objective lens 5, an optical path switching mirror 7, an
eyepiece 8, a camera lens 9, a camera 10 serving as an image pickup
device, and an autofocus optical system composed of an autofocus
light source portion 11, a half mirror 12, a dichroic mirror 13, an
autofocus light-collecting portion 14, and an autofocus
light-receiving portion 15.
[0037] The observation optical system is configured such that an
optical image of the observation target object 1 that has been
obtained with the objective lens 5 can be observed as a still image
or a moving image by visual inspection or by the use of the camera
10 serving as the image pickup device. Although the optical
microscope of the present exemplary configuration is configured to
switch between a mode in which the optical path switching mirror 7
is disposed in the observation optical system and visual inspection
is performed via the eyepiece 8 and a mode in which image data is
obtained with the camera 10 via the camera lens 9, it is also
possible to adopt a configuration in which the visual inspection
and the acquisition of image data can be performed
simultaneously.
[0038] The camera 10 is an image pickup device including a CCD
(Charge Coupled Device) image sensor as an image pickup element. As
will be described later, the camera 10 of the optical microscope
according to the present exemplary configuration is configured to
alternately repeat two operation periods, namely, a capturing
period during which a large number of light-receiving elements each
receive, as an optical signal, an optical image of the observation
target object 1 that has been obtained through the camera lens 9 by
the objective lens 5, and a readout time during which the electric
charge obtained by each of the light-receiving elements is sent out
as an electric signal, and the camera 10 is capable of converting
an optical signal into an electric signal and sending out the
signal. In addition, the camera 10 transmits the operation timing
between the readout time and the capturing period of the operation
periods to the control portion 31 of the optical microscope.
[0039] The captured image of the observation target object 1
obtained with the camera 10 is sent out as an electric signal to a
monitor 32 that is attached to the optical microscope or disposed
separately as an external device, and an enlarged image of the
observation target object 1 can be observed in real time on the
monitor 32. Note that the monitor 32 may serve as a monitor portion
of the computer as shown in FIG. 1 and display an image obtained
after performing image processing on the obtained image signal on a
computer, or may be configured as a display portion physically
integrated with and attached to the optical microscope, and having
only the function of directly displaying an image of the
observation target object 1.
[0040] The autofocus light source portion 11 of the autofocus
optical system is made up of a focusing light source 16 composed
of, for example, an infrared LED that emits near-infrared light, a
slit 17, a collimating lens 18, a pupil-limiting portion (not
shown), and the like, and emits autofocus light.
[0041] The autofocus light emitted from the autofocus light source
portion 11 enters the objective lens 5 via the half mirror 12 and
the dichroic mirror 13 that reflects red light, and is collected at
the interface portion between the slide glass 2 and the cover glass
4 that are located at the same planar position as the observation
target object 1. The autofocus light that has been reflected at the
interface portion of the slide glass 2 or the cover glass 4, or at
a reflection film, which may be provided so as to perform more
accurate autofocus, passes through the dichroic mirror 13 and the
half mirror 12, and enters the autofocus light-collecting portion
14.
[0042] The autofocus light that has entered the autofocus
light-collecting portion 14 is collected at the autofocus objective
lens 19, temporarily imaged as a slit image by the image-forming
portion 20 including a slit portion and a relay lens, passes
through the cylindrical lens 21, and is imaged again in the
autofocus light-receiving portion 15.
[0043] The autofocus light-receiving portion 15 is configured to
convert an optical signal into an electric signal by using a
light-receiving element such as a CCD image sensor as with the
camera portion 10, and it detects the imaged slit image of the
autofocus light as an electric signal, and transmits the focus
state to the control portion 31.
[0044] The control portion 31 determines the focus state at the
objective lens 5 based on the state of the slit image of the
autofocus light transmitted from the autofocus light-receiving
portion 15, and performs control to constantly maintain the focus
state by correcting the focal position of the objective lens 5 if
there has been a shift in focus. As the method for correcting the
focal position of the objective lens 5, it is possible to use
various optically utilizable methods, including, for example, a
method of moving the placement position of the objective lens 5
itself along the optical axis, and a method of varying the
intervals between lenses if the objective lens 5 is configured as a
group of a plurality of lens.
[0045] Thus, in the optical microscope shown in FIG. 1, the
autofocus optical system, the control portion 31, the objective
lens 5, and the adjustment mechanism of the focal position thereof
constitute the autofocus device for the optical microscope.
[0046] Note that the components of the observation optical system
of an ordinary optical microscope can be directly applied to the
observation optical system of the optical microscope in the present
exemplary configuration described above. Therefore, although not
shown in FIG. 1, an irradiation light source can be disposed so as
to irradiate the observation target object 1, from below the slide
glass 3 in the case of the transmission type, or from above the
slide glass 3 via the objective lens 5 through a part of the
observation optical system in the case of the epi-illumination
type. Although the objective lens 5 is illustrated as a single lens
in FIG. 1, a plurality of objective lenses having different
magnifications can be disposed so as to be switchable using a
revolver. By placing a motor for rotating the revolver, the
objective lenses can be made electrically switchable via a signal
from the control portion 31, making it possible to vary the
observation magnifications.
[0047] As with the observation optical system, the autofocus
optical system of the optical microscope according to the present
exemplary configuration described above can use the components of
an autofocus optical system based on an ordinary activefocus
method. Also, a commonly used control can be used for the control
content for performing control to determine the focus state based
on the image data obtained with the autofocus light-receiving
portion 14, and to correct the focus position of the objective lens
5 so as to maintain the focus state. Therefore, the autofocus
optical system of the optical microscope according to the present
embodiment is not limited to the exemplary configuration shown and
described above.
[0048] FIG. 2 shows a relationship between the exposure timing in
each image pickup element in the camera 10 of the observation
optical system and the irradiation timing of autofocus light in the
focus light source 16 of the autofocus optical system in the
optical microscope according to the present embodiment.
[0049] As shown in FIG. 2, in the optical microscope of the present
embodiment, autofocus light is emitted during the readout time of
electric signals in the CCD image sensor used in the camera 10.
[0050] In general, when the CCD image sensor is used as an image
pickup element, the electric charges stored in each of the
light-receiving elements during the capturing period are read to
the outside via a transfer electrode during the readout time at a
predetermined timing. Since the electric charges in all the
light-receiving elements are sequentially transferred and read out
during the readout time, the light-receiving elements do not store
an electric charge during the readout time, and any optical image
obtained with the objective lens 5 will not be converted into an
electric signal during this period. Accordingly, even if autofocus
light is emitted to the observation target object 1 during the
readout time, no adverse effect will be exerted on a captured image
obtained with the camera 10. By ending the irradiation timing of
autofocus light within the readout time of the camera 10 in the
above-described focus operation based on a focused image obtained
with the autofocus optical system, the image data for the capturing
period during which autofocus light is not emitted can be obtained
with the camera 10.
[0051] Note that some cameras may allow the length of the readout
time to be adjusted by the user, and in that case, it is possible
to achieve a more favorable image observation of the observation
target object by setting the shortest possible readout time within
the range that the camera does not receive autofocus light, taking
into consideration the irradiation time of autofocus light and the
time required for the autofocus operation in the autofocus device
included in the optical microscope.
[0052] In the optical microscope of the present embodiment, the
irradiation time of autofocus light is set to 100 .mu.s as an
example. Further, since the time required for the autofocus
operation can be made very short by using means having a high-speed
tracking performance such as a piezoelectric element or a stepping
motor as the means for adjusting the focal position of the
objective lens, the autofocus operation can be completed during the
non-capturing period of the camera 10 in the case of the optical
microscope of the present embodiment. Note that the irradiation
timing of the autofocus light may be set to any timing during the
readout time as long as the irradiation ends within the readout
time.
[0053] Note that in the case of capturing a moving image of the
observation target object 1, captured images during the capturing
period are continuously obtained while the capturing period and the
non-capturing period are repeated in the camera 10 serving as the
image pickup device. In this case as well, captured images in a
correct focus state can be constantly obtained by performing the
autofocus operation during the non-capturing period. In particular,
a shift in focus, which is called "focal drift", due to a change in
the environment tends to occur in the case of a long-time image
capturing. However, with the optical microscope of the present
disclosure, it is possible to obtain a favorable moving image by
tracking this focal drift.
[0054] Although a plurality of capturing periods and non-capturing
periods alternately appear in this case, the focus operation can be
performed in all of the plurality of non-capturing periods, and the
focus operation can be performed in any selected non-capturing
period among the plurality of non-capturing periods. For example,
when it is known in advance that the amount of focal drift during a
long image capturing time is small, the frequency with which
autofocus light is emitted to the observation target object 1 is
reduced by thinning out the timings of performing the autofocus
operation so as to be limited to the selected non-capturing
periods. As a result, even if there is any stimulus to the
observation target object 1 applied by the autofocus light or any
undesired entry of the autofocus light into a captured image, these
can be effectively reduced. Note that the timing of performing the
autofocus operation may be set at regular intervals by setting the
timing for every predetermined number of non-capturing periods, or
may be set at irregular intervals by randomly selecting the
non-capturing periods in which the autofocus operation is
performed.
[0055] As described above, with the optical microscope of the
present embodiment, the autofocus light is emitted during the
non-capturing period in the image pickup device, and thereby, image
data of the observation target object that is free from the
influence of the autofocus light can be obtained even in the case
where image capturing with a highly sensitive detector or long-time
image capturing is required because of a weak emission luminance of
the observation target object, as in chemiluminescence observation,
for example.
[0056] FIG. 3 is a block diagram for illustrating a schematic
configuration in an inverted configuration in which the objective
lens is disposed above the observation target object, as a second
exemplary configuration of the optical microscope according to the
present embodiment.
[0057] In the optical microscope of the second exemplary
configuration according to the present embodiment shown in FIG. 3,
an observation target object 51 is, for example, the nucleus of a
cell serving as a sample 52, and the cell serving as the sample 52
is disposed in a nutrient solution 54 in a petri dish 53. The petri
dish 53 is placed on a cover glass 55 as a glass substrate also
serving as a platform, and an interface 56 is formed between the
petri dish 53 and the cover glass 55.
[0058] The cover glass 55 is disposed above the objective lens
device 57 via a predetermined optical coupling member 58. Note that
the optical axis of an objective lens (not shown) in the objective
lens device 57 may be movable relative to the observation target
object 51.
[0059] The optical microscope of the second exemplary configuration
includes an observation optical system composed of the objective
lens in the objective lens device 57, a first mirror 58, a half
mirror 59, a second mirror 60, an eyepiece 61, a camera lens 62,
and a camera 63 serving as an image pickup device, and an autofocus
optical system composed of an autofocus light source portion 71, a
half mirror 72, a focus optical path adjustment portion 73, a
dichroic mirror 74, an autofocus light-collecting portion 75, and
an autofocus light-receiving portion 76.
[0060] The observation optical system is configured such that an
optical image of the observation target object 51 that has been
obtained with the objective lens in the objective lens device 57
can be observed as a still image or a moving image by visual
inspection or by the use of the camera 63. With the optical
microscope of the present exemplary configuration, the visual
inspection via the eyepiece 61 with the half mirror 59 disposed in
the observation optical system and the acquisition of image data by
the camera 63 via the camera lens 62 can be performed
simultaneously.
[0061] The camera 63 is an image pickup device including a CCD
image sensor as an image pickup element. As shown in FIG. 2, the
camera 63 of the optical microscope of the present exemplary
configuration also has, as exposure timings, two operation periods,
namely, a capturing period during which a large number of
light-receiving elements each receive an optical image of the
observation target object 51, and a readout time during which an
electric signal such as an electric charge obtained with each of
the image pickup elements is sent to the outside. In addition, the
camera 63 transmits the operation timing between the capturing
period and the capturing period during the exposure timing to the
control portion 91 of the optical microscope.
[0062] The captured image of the observation target object 51
obtained with the camera 63 is sent as an electric signal, for
example, to a computer 92, which is an external device, and is
displayed on the computer monitor after being subjected to the
desired image processing. Note that as with the optical microscope
of the first exemplary configuration described with reference to
FIG. 1, the data of the captured image obtained with the camera
portion 63 can be displayed on the monitor portion attached to the
optical microscope.
[0063] As with the first exemplary configuration shown in FIG. 1,
the autofocus light source portion 71 of the autofocus optical
system is made up of, for example, a focusing light source 77
composed of an infrared LED that emits near-infrared light, a slit
78, a collimating lens 79, a pupil-limiting portion (not shown),
and the like, and autofocus light is emitted during the readout
time, which is a non-capturing period, of the camera portion
63.
[0064] The autofocus light that has been emitted from the autofocus
light source portion 71 enters the objective lens of the objective
lens device 57 via the half mirror 72, the focus optical path
adjustment portion 73, and the dichroic mirror 74 that reflects red
light, and is collected at the interface 56 between the petri dish
53 and the cover glass 54. The autofocus light that has been
reflected at the interface 56 between the petri dish 53 and the
cover glass 54 passes through the dichroic mirror 74, the focus
optical path adjustment portion 73, and the half mirror 72, and
enters the autofocus light-collecting portion 75.
[0065] The autofocus light that has entered the autofocus
light-collecting portion 75 is collected at the autofocus objective
lens 80, temporarily imaged as a slit image by the image-forming
portion 81 including a slit portion and a relay lens, passes
through the cylindrical lens 82, and is imaged again in the
autofocus light-receiving portion 76.
[0066] The autofocus light-receiving portion 76 is configured to
convert an optical signal into an electric signal by a
light-receiving element such as a CCD image sensor, and it detects
the imaged slit image of the autofocus light as an electric signal,
and transmits the focus state to the control portion 91.
[0067] The control portion 91 determines the focus state at the
objective lens device 57 based on the state of the slit image of
the autofocus light transmitted from the autofocus light-receiving
portion 76, and performs control to maintain the focus state by
correcting the focal position of the objective lens by adjusting
the objective lens device if there has been a shift in focus. Note
that the optical microscope of the second exemplary configuration
includes the focus adjustment lens 73 in the focus optical system
as with the autofocus device disclosed in Patent Document 2, and is
configured such that a predetermined focus offset amount is set for
the autofocus light reflected at the interface 56 between the petri
dish 53 and the cover glass 54, thereby making it possible to
maintain the focus state to the observation target object 51, which
is located at a predetermined interval from the interface 56, which
is an actual reflection plane of the autofocus light.
[0068] In the optical microscope of the second exemplary
configuration shown in FIG. 3 as well, the control portion 91
controls the readout timing of image signals from the image pickup
elements in the camera 63 of the observation optical system and the
irradiation timing of the autofocus light in the focus light source
77 of the autofocus optical system such that the autofocus light is
emitted during the readout time of electric signals from the
light-receiving elements of the camera portion 63 as shown in FIG.
2. Accordingly, in the optical microscope of the second exemplary
configuration shown in FIG. 3 as well, the image data of the
observation target object 51 for only a period during which the
autofocus light is not applied can be obtained with the camera
portion 63 by ending the irradiation with the autofocus light
within the readout time of the image pickup element of the camera
portion 63 in a focus operation based on a focused image obtained
with the autofocus optical system.
[0069] Note that in the inverted second exemplary configuration
shown in FIG. 3, conventionally known configurations can be used as
the observation optical system and the autofocus optical system, as
with the first exemplary configuration shown in FIG. 1. Therefore,
the configuration shown in FIG. 3 is merely an exemplary
configuration. In the optical microscope of the second exemplary
configuration shown in FIG. 3 as well, an irradiation light source
(not shown) can be disposed so as to irradiate the observation
target object 51 via a part of the observation optical system.
Further, in the objective lens device 57 shown in FIG. 3, a
plurality of objective lenses having different magnifications can
be disposed so as to be automatically switchable. Conversely, by
including the focus adjustment lens used in the second exemplary
configuration described with reference to FIG. 3 in the autofocus
optical system, a predetermined focus offset amount can also be set
for the autofocus light in the optical microscope of the first
exemplary configuration described with reference to FIG. 1.
[0070] As described thus far, with the optical microscope of the
present embodiment, the irradiation timing of the autofocus light,
which is emitted for performing autofocus, from the focus light
source is controlled such that the autofocus light is emitted
during the non-capturing period during which image data is not
captured in the image pickup device that acquires image data of the
observation target object. By doing so, it is possible to reliably
avoid the effect of the autofocus light from exerted on a captured
image of the observation target object that is captured by the
image pickup device.
[0071] FIG. 4 show a comparison between a fluorescence image of a
HeLa cell that expresses a fluorescent protein in a cell membrane
(FIG. 4(a)) and a chemiluminescence image of the same cell that
expresses a chemiluminescent protein in a cell membrane (FIG. 4
(b)), each obtained with a conventional optical microscope. Note
that an objective lens with a magnification of 60.times. is used.
As shown in FIG. 4, the cell configuration is hardly observed by
the chemiluminescence observation because of a small luminance of
the observation target object.
[0072] In contrast, FIG. 5 show a fluorescence observation image
(FIG. 5(a)) and a chemiluminescence observation image (FIG. 5 (b))
of mitochondria, obtained with the luminescent protein Nano-lantern
whose luminance has been increased by high-efficiency FRET from
that of a chemiluminescent protein to that of a high-luminance
fluorescent protein. Note that an objective lens with a
magnification of 60.times. is used.
[0073] As shown in FIG. 5, a clear observation image equivalent to
that obtained by fluorescence observation was also obtained by
chemiluminescence observation. Accordingly, it is possible to
perform a more favorable cell observation as compared with
fluorescence observation, which may cause damage to a cell to be
observed because it requires irradiation with ultraviolet light,
which is excitation light. Nano-lantern is a high-luminance
chemiluminescent probe, and thus enables video-rate observation.
Therefore, combining this probe with the optical microscope of the
present embodiment makes it possible to perform a high-speed cell
observation without concern for a focus shift due to a temperature
change or stage variations.
[0074] Note that the provision of a configuration that emits
excitation light for performing fluorescence observation makes it
possible to use the optical microscope described in the present
embodiment for fluorescence observation. Since the autofocus
irradiation light described in the embodiment above has an
irradiation energy sufficiently smaller than an irradiation energy
of 0.1 to 1 W/cm.sup.2, which is required to stimulate a cell to be
observed, there is no possibility that a cell to be observed causes
any undesired reaction by being irradiated with the autofocus
light.
[0075] Furthermore, with the optical microscope according to the
present disclosure, the timing of emitting the autofocus light is
different from the acquisition timing of image data. Accordingly,
the autofocus light is not limited to light outside the visible
range, and it is also possible to use visible light. Therefore, it
is possible to use, as the autofocus light, light of the desired
wavelength that allows autofocus to be performed accurately and in
a short time, making it possible to improve the precision of
autofocus. In addition, when the amount of light emitted by the
observation target object is small as in the case of
chemiluminescence observation, it is conceivable that a dichroic
mirror having a higher transmittance is used as the dichroic mirror
disposed in the observation optical system for emitting the
autofocus light, thereby detecting the emission of the observation
target object with as little loss of light as possible.
[0076] Moreover, for the optical microscope of the present
embodiment described above, an example is disclosed in which a CCD
image sensor is used as the image pickup device. However, it is
possible to use an image pickup device including a semiconductor
image pickup element such as a CMOS image sensor. In the case of a
CMOS image sensor, the readout time of reading out the electric
charges stored in the light-receiving elements at a time is not
provided as in the case of the CCD image sensor, and readout is
performed in the line sequential method. However, the so-called
blank period corresponds to the non-capturing period, and
therefore, autofocus light can be applied during the non-capturing
period. Furthermore, the image pickup device used for the optical
microscope of the present embodiment is not limited to those
including these semiconductor image pickup elements. For example,
it is possible to use an image pickup device including a vacuum
tube-type image pickup element such as a photomultiplier and any of
various other types of image pickup elements. In this case,
depending on the image pickup device, it may not be necessary to
provide the non-capturing period such as the CCD readout time in
the operation periods. In that case, a non-capturing period may be
forcibly formed in the operation periods, and autofocus light may
be emitted in timing with that non-capturing period.
INDUSTRIAL APPLICABILITY
[0077] The optical microscope and the autofocus device for an
optical microscope disclosed in the present application are
expected to be used in a wide variety of fields as an optical
microscope capable of performing imaging by various methods,
including, for example, chemiluminescence observation, and an
autofocus device for the optical microscope.
* * * * *